Exploring ND-011992, a quinazoline-type inhibitor targeting quinone reductases and quinol oxidases

Bacterial energy metabolism has become a promising target for next-generation tuberculosis chemotherapy. One strategy to hamper ATP production is to inhibit the respiratory oxidases. The respiratory chain of Mycobacterium tuberculosis comprises a cytochrome bcc:aa3 and a cytochrome bd ubiquinol oxidase that require a combined approach to block their activity. A quinazoline-type compound called ND-011992 has previously been reported to ineffectively inhibit bd oxidases, but to act bactericidal in combination with inhibitors of cytochrome bcc:aa3 oxidase. Due to the structural similarity of ND-011992 to quinazoline-type inhibitors of respiratory complex I, we suspected that this compound is also capable of blocking other respiratory chain complexes. Here, we synthesized ND-011992 and a bromine derivative to study their effect on the respiratory chain complexes of Escherichia coli. And indeed, ND-011992 was found to inhibit respiratory complex I and bo3 oxidase in addition to bd-I and bd-II oxidases. The IC50 values are all in the low micromolar range, with inhibition of complex I providing the lowest value with an IC50 of 0.12 µM. Thus, ND-011992 acts on both, quinone reductases and quinol oxidases and could be very well suited to regulate the activity of the entire respiratory chain.

Tuberculosis caused by Mycobacterium tuberculosis is one of the infectious diseases with the most causes of death worldwide mainly affecting developing countries 1 . With the emergence of COVID-19, tuberculosis was briefly replaced as the leading cause of death from a single infectious disease, but will again play a major role in the future. Since conventional medication is showing less and less success due to the development of multi-drug (MDR) and extensively drug-resistance (XDR) 2 , there is an urgent need for new medications. The M. tuberculosis enzyme complexes involved in energy metabolism that synthesise the vast majority of ATP have recently come into the focus of interest for the development of new antibiotics [3][4][5] . A major difficulty in controlling tuberculosis is that M. tuberculosis can enter a non-replicating phase 6 when exposed to stresses, such as the lack of oxygen, and can persist in this phase for a very long time. Important for this survival mechanism are the enzyme complexes responsible for energy metabolism such as bd oxidase, which is up-regulated under these conditions 7 . The respiratory chain of M. tuberculosis comprises two terminal oxidases 8 . Under high oxygen tension the cytochrome bcc:aa 3 oxidase is produced, while at low oxygen conditions the bd oxidase is active. Previous studies showed that inhibition of both M. tuberculosis terminal oxidases resulted in an effective growth suppression of antibiotictolerant mycobacteria 9 . Inhibition of cytochrome bcc:aa 3 oxidase is achieved by the drug candidate Telacebec (Q203) that has already been tested in a phase 2a clinical efficacy trial 10 . To prevent M. tuberculosis from using the alternative route for electron transfer maintaining respiration and thus ATP synthesis 11 , the cytochrome bd oxidase has to be inhibited in combination with the cytochrome bcc:aa 3 oxidase. Recently, a small organic compound N-(4-(4-(trifluoromethyl)phenoxy)phenyl)quinazolin-4-amine (also called ND-011992) was identified that was shown to inhibit cytochrome bd oxidase 9 . However, the potential use of ND-011992 as anti-tuberculosis drug is hampered by its poor pharmacokinetic properties as already mentioned in the original publication 9 . Thus, optimization of the lead structure is urgently needed to improve the inhibitory potential and pharmacokinetics of ND-011992 8,9 . Scientific Reports | (2023) 13:12226 | https://doi.org/10.1038/s41598-023-39430-w www.nature.com/scientificreports/ Furthermore, a similar quinazoline-based inhibitor 4-N-[2-(4-phenoxyphenyl)ethyl]-quinazolin-4,6-diamine (EVP4593) was reported by one of us to inhibit mitochondrial complex I, another important enzyme complex of the respiratory chain 12 . The common aromatic core structure of ND-011992 and EVP4593 opened up the question of whether ND-011992 is specific for bd oxidase or whether it could also inhibit complex I (Fig. 1).
In this work, the inhibition of complex I as well as the bo 3 and bd terminal oxidases of the respiratory chain in Escherichia coli by ND-011992 and its bromine containing derivative, compound 2 ( Fig. 1) were investigated. Compound 2 has an elevated clogP value, signifying heightened lipophilicity and a closer resemblance to the clogP of established drug candidate Telacebec when compared to compound 1. Moreover, the motivation behind incorporating a halogenated derivative stemmed from the possibility that if compound 1 demonstrated good binding to one of the enzymes under investigation, the brominated compound 2 would be well suited to localize the binding site on this enzyme via cryo-electron microscopy. To this end, we are planning on conducting extensive structure-activity-relationship studies in our next work. The aforementioned enzymes are very well suited for this study, since the E. coli bd oxidases show a high structural similarity to the M. tuberculosis bd oxidase 8 . Furthermore, E. coli respiratory complex I is a structural minimal form of mitochondrial complex I. Our studies reveal that ND-011992 inhibits quinone reducing complex I and quinol oxidizing oxidases.

Results
Synthesis of ND-011992 and its derivative. ND-011992 (1) and a ND-011992 derivative (2) with a bromine substituting for H at position 6 ( Fig. 2) were synthesized in a one-step reaction modified from a procedure by Lee et al. 9 as described in the 'Methods' section.
4-Chloroquinazoline or 6-Bromo-4-chloroquinazoline, respectively, were reacted with 4-(4-(trifluoromethyl) phenoxy)aniline and potassium carbonate in DMSO. All starting materials are commercially available and the reaction affords products in moderate yields (75% for 1 and 45% for 2). Both crude compounds were purified via silica gel flash chromatography. Chlorine and iodine derivatives of this compound (not further described in this work) were synthesized in analogous manner. Compounds were characterized via 1 H-and 13 C-NMR as well as via MS (see Supplementary Figs. S1-S6).  www.nature.com/scientificreports/ This reaction constitutes a nucleophilic aromatic substitution (S N Ar) reaction, where the chlorine on the quinazoline reactant acts as a leaving group that is displaced by the nucleophilic aryl amine reactant. The proposed reaction mechanism is shown in Fig. 3.

Inhibition of membrane bound enzyme complexes.
The inhibitory effect of ND-011992 on the NADH oxidase activity of membranes from E. coli strain BL21*Δcyo was determined. This activity is mainly mediated by the primary NADH dehydrogenases and the terminal oxidases 13 . Strain BL21*Δcyo chromosomally lacks the bo 3 -oxidase and contains the homologous bd-I and bd-II oxidases as the only terminal oxidases 14 . BL21*Δcyo was grown in full medium, cells were harvested and disrupted and cytoplasmic membranes were prepared by differential centrifugation. With NADH as substrate, electrons might enter the respiratory chain either via complex I or via the alternative, non-energy converting NADH dehydrogenase, NDH-II. Titration of the NADH oxidase activity with ND-011992 revealed an IC 50 of 0.43 µM (Fig. 4a). Second, deamino-NADH (d-NADH) was used as substrate to determine the d-NADH oxidase activity of BL21*Δcyo membranes. d-NADH is a substrate specific for complex I preventing electron entry to the respiratory chain via NDH-II 15,16 . In this case, titration with ND-011992 revealed a significant lower IC 50 of 0.26 µM (Fig. 4b). The shift of the IC 50 in dependence from the substrate already indicates a high-affinity inhibition of respiratory complex I by ND-011992. To specifically monitor the inhibition of complex I, the d-NADH:decyl-ubiquinone oxidoreductase activity of cytoplasmic membranes of strain CBO was measured.
Strain CBO is chromosomally lacking both bd oxidases leaving bo 3 oxidase as the only terminal oxidase 17 . The activity of bo 3 oxidase was completely inhibited by an addition of 1 mM KCN to the assay 18 . Decyl-ubiquinone was added as external electron acceptor collecting electrons from the quinone-pool that is reduced by the reaction of complex I. With d-NADH as substrate, complex I is the only active enzyme in the assay. Here, the IC 50 towards ND-011992 was determined to 0.12 µM (Fig. 4c). Thus, ND-011992 exhibits a sub-micromolar affinity towards complex I, making it a good inhibitor of this enzyme. The two-fold difference in the IC 50 of ND-011992 in the d-NADH oxidase activities ( Fig. 4b and c) might be due to differences between the two E. coli strains or to the additional contribution of the bd oxidases to the d-NADH oxidase activity. It can be concluded that ND-011992 is a good inhibitor of respiratory complex I in the bacterial membrane with an IC 50 of 0.12 µM. To validate the claim that ND-011992 is a good and specific inhibitor of bd oxidases, we next investigated its inhibitory effect on the isolated oxidases.
The affinity of ND-011992 towards the isolated oxidases was determined by its inhibition of the duroquinol:oxygen oxidoreductase activity measured as decrease of oxygen concentration 20 . The IC 50 to bd-I and bd-II oxidase were determined to 0.63 and 1.3 µM, respectively ( Fig. 5a and b). Thus, there is a significant two-fold difference in binding of ND-011992 towards the two homologous oxidases. Furthermore, the IC 50 is higher than that to respiratory complex I, showing that the compound binds better to complex I than to bd-I and to bd-II oxidase. Incorporation of bromine to the compound led to significant changes of inhibition. The IC 50 of the bromine derivative 2 to bd-I oxidase was slightly increased to 1.03 µM compared to that obtained with www.nature.com/scientificreports/ ND-011992. In contrast, the brominated compound showed a tenfold improved binding to bd-II oxidase with an IC 50 of 0.13 µM ( Fig. 5c and d). Surprisingly, 2 had no effect on the NADH oxidase activity of E. coli membranes in the micromolar concentration range. The most reasonable explanation is that 2 cannot enter the biological membrane due to the bulky bromine substitution and therefore does not encounter its target.
Because it was claimed that ND-011992 is an inhibitor specific for bd oxidases 9 , we also determined its IC 50 towards the isolated bo 3 oxidase. As bd oxidases, bo 3 oxidase is also a quinol oxidase. Indeed, the IC 50 of 2.47 µM is two-to four-fold higher than for the bd oxidases (Fig. 5e), however, all IC 50 values are in a similar range.
In these experiments, all three oxidases are only partially inhibited, especially in the titration with ND-011992 about 20% residual activity remains (Fig. 5). Partial inhibition is found when an enzyme-inhibitor complex is still capable of producing a product. This clearly indicates that ND-011992 acts as a reversible inhibitor that does not bind to the exactly same site as the substrate but in close proximity. This is expected due to the distinctly different structures of the inhibitors and the substrate. In addition, the quinone binding sites of all the enzymes investigated have a completely different architecture (see 'Discussion'), so that competitive inhibition to the respective substrate binding site is not to be expected.
Inhibition of E. coli respiratory complex I by ND-011992 at sub-micromolar concentrations was unexpected. In order for ND-011992 or a derivative thereof to be used as a possible anti-tuberculosis drug, it must be ensured that the substance does not also inhibit mitochondrial complex I. To verify this, we investigated the inhibition of NADH oxidase activity of membranes from bovine heart mitochondria (Fig. 6). The rate-limiting step of this reaction cascade is respiratory complex I. The NADH oxidase activity was inhibited with an IC 50 of 3.27 µM. Again, a residual activity of about 20% is observed. Inhibition of mitochondrial complex I is about 27-fold lower than that of bacterial complex I. This shows that ND-011992 is a suitable starting material to develop more effective and specific derivatives that specifically inhibit bacterial bd oxidases.

Discussion
We investigated the effect of ND-011992, which has been described as an inhibitor of bd oxidases 9 , on various enzyme complexes of the E. coli aerobic respiratory chain. The IC 50 values of ND-011992 to respiratory complex I, bo 3 oxidase and both E. coli bd oxidases are all in the low micromolar range. Thus, ND-011992 is not a specific inhibitor that acts with high affinity on one target, but it inhibits quinone reductases and quinol oxidases of the respiratory chain. These enzyme complexes all have distinct differences in the architecture of their quinone The aurachin polar head group interacts with a conserved Asp residue and its hydrophobic chain is involved in several hydrophobic interactions with residues of the Q-loop and the transmembranous region of the same subunit. In contrast, the ubiquinone reduction site of respiratory complex I is made up by several subunits that form a large cavity extending from the hydrophobic membrane into the so-called peripheral arm of the complex I containing the cofactors of electron transfer. The ubiquinone headgroup is bound by conserved Tyr and His residues buried deep at the end of the cavity [28][29][30] . In the E. coli complex I, the isoprene side chain of ubiquinone-8 fills the entire cavity and faces several hydrophobic interactions 28 . Due to the different structural arrangement of the quinone binding sites, it is reasonable to assume that ND-011992 mainly acts through its hydrophobicity and space filling structure to bind and block the access to the active sites. Remarkably, ND-011992 inhibits quinone reduction as found in respiratory complex I, and quinol oxidation, as present in quinol oxidases with similar efficacy. Thus, the compound exhibits a broad spectrum of putative targets. This makes it an interesting tool to e.g. efficiently shut down the entire respiratory chain. On the other hand it can be used as a starting point to develop more specific inhibitors. The molecule is freely available from one of the authors (DL) upon reasonable request.
Nevertheless, subtle differences were detectable in the inhibition of the various enzyme complexes of the respiratory chain. ND-011992 shows the best inhibitory power towards complex I with an IC 50 of 0.12 µM (Fig. 4). The bd oxidases are inhibited with a five to eleven times lower efficacy. Remarkably, the IC 50 to bd-I (0.63 µM) is twofold lower than to bd-II (1.3 µM). Furthermore, the bromine derivative 2 has a tenfold lower IC 50 (0.13 µM) to bd-II than ND-011992 (Fig. 5). This might be due to the presence of a positively charged Lys residue in the large subunit of bd-II oxidase (K287) that is replaced by a neutral Gln residue in bd-I (Q287) in the putative Q-binding side of the bd oxidases. This makes this compound an attractive tool to study the quinol binding site of bd-II oxidase by cryo-electron microscopy.
Most interestingly, mitochondrial complex I is inhibited with a 27 times lower efficacy than E. coli complex I (IC 50 = 0.12 vs. 3.27 µM). Furthermore, the inhibition of bacterial bd oxidases, which have no homologues in mitochondria, is still five to three times more effective than that of the mitochondrial complex. Thus, there is a good chance that further structural optimization of ND-011992 will lead to the development of even more specific and potent inhibitors. Lead optimization is also needed to improve the not so outstanding pharmacokinetic properties of ND-011992.
In light of the inhibitory results for ND-011992 (1) and bromine derivative 2 we conclude that these two molecules can be considered novel chemical probes for quinone/quinole converting enzymes due to their ability to entirely shut down respiration. With this work we are aiming to contribute to "target 2035", an ambitious initiative by academic and pharmaceutical groups to develop small molecule probes for every protein in the proteome 31 .

Methods
General experimental and instrumentation for chemical synthesis. Commercially available reagents were purchased from Alfa Aesar, Millipore Sigma, and Acros Organics. Reaction setup included ovendried glassware, magnetic stir plate and stir bar, a thermometer, and a heating mantle. Reactions were monitored by Thin-layer chromatography using Analtech Silica gel HLF UV254. TLC plates were visualized using a UV lamp. Flash chromatography was done with Acros Organics 0.035-0.070mm, 60 Å silica gel as the stationary phase. ClogP data were obtained using the chemical properties tool in ChemDraw.
1 H-and 13 C-NMR data were taken on an Agilent VnmrJ4 400 MHz NMR Spectrometer and recorded in ppm against the NMR solvent (CDCl 3 ) or TMS as the internal standard. Spectral data are reported in the following format: (1) 1 H-NMR spectroscopy: chemical shifts (multiplicity, coupling constant, and number of protons); (2) 13 C-NMR spectroscopy: chemical shift. Chemical shifts (δ) were reported in parts per million (ppm) and determined relative to the internal standard TMS (δ 0.0 ppm). Multiplicities were abbreviated as the follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublets of doublet, m = multiplet, br = broad. GC/MS data were obtained using Agilent 6890N/5973 GCMS system. -011992 (1)). 4 (2)). 6 Strains and cell growth. Cytoplasmic membranes from E. coli strain BL21*Δcyo that chromosomally lacks the cyoABCD genes encoding cytochrome bo 3 oxidase and strain CBO that chromosomally lacks the cydABX and appCBX genes encoding the cytochrome bd-I and bd-II oxidase, respectively, were used to determine enzyme activity in the membrane 13,17,19 . Cytochrome bo 3 ubiquinol oxidase was isolated from E. coli strain BL21*Δcyo/ pET28b( +)::cyoA his BCD with the genes encoding the oxidase on an expression plasmid. Cytochrome bd-I ubiquinol oxidase was isolated from strain BL21*Δcyo/pET28b( +)::cydA his BX with the genes encoding the bd-I oxidase on an expression plasmid 14 . Cytochrome bd-II ubiquinol oxidase was isolated from strain BL21*Δcyo/ pET28b( +)::appC his BX with the genes encoding the bd-II oxidase on an expression plasmid 19 . Cell cultures were grown aerobically in 2 L baffled flasks each containing 800 mL LB-medium at 37 °C. Gene expression was induced at an OD 600 of approximately 2 by the addition of 400 µM isopropyl β-D-1-thiogalacto-pyranoside. Cells were harvested 2 h after induction by centrifugation and stored at -80 °C 19 .

Synthesis of 6-bromo-N-(4-(4-(trifluoromethyl)phenoxy)phenyl)quinazolin-4-amine (also referred to as bromine derivative
Preparation of cytoplasmic membranes. All steps were carried out at 4 °C. 40 g shock frozen cells (wet weight) were homogenized in a six-fold volume of 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 20 mM NaCl, 0.5 mM phenylmethylsulfonylfluoride (PMSF), pH 7.0 and a few grains of DNase I in a teflon-inglass homogenizer. Cells were disrupted with three cycles in a high pressure homogenizer (1250 bar, Maximator HPL6, Maximator GmbH) and cell debris was removed by centrifugation (4 °C, 10′000 rpm, 30′000 × g; 20 min, Rotor 25.50, Avanti J-26S XP). Membranes were obtained from the supernatant by ultra-centrifugation (4 °C, 50′000 rpm, 250′000 × g, 70 min, Rotor 60Ti, Optima LE80-K, Beckman-Coulter). The sedimented membranes were re-suspended in a 1:1 ratio (w/v) with 20 mM MOPS and 20 mM NaCl, pH 7.0 in a teflon-in-glass homogenizer.  19 . 5 µL membrane suspensions (50 mg/mL) were added to 2 mL 20 mM MOPS, 20 mM NaCl, pH 7.0. The non-enzymatic reaction was determined after formation of a stable baseline. The reaction was started by an addition of 5 µL (d-)NADH (0.5 M) and the catalytic rates were corrected for the non-enzymatic reaction. After 1 min, the reaction was inhibited by adding various amounts (0.01-50 µM) ND-011992. Each data point was assayed in triplicates. The protein concentration of membranes were determined by the biuret-method 32 . The standard deviation of each measurement is provided. The d-NADH:decyl-ubiquinone oxidoreductase activity of cytoplasmic membranes was measured as decrease of the d-NADH concentration at 340 nm using an ε of 6.3 mM −1 cm −1 (QS cuvette, d = 1 cm, Hellma; TIDAS S500, J&M Aalen) 15 . 10 µL membrane suspension (100 mg/mL) were added to 1 mL 50 mM MES/NaOH, 50 mM NaCl, 5 mM MgCl 2 , pH 6.0. The activity of the bo 3 oxidase was inhibited by an addition of 1 mM KCN. 150 μM d-NADH was added after 30 s incubation. The reaction was started after 1 min by an addition of 400 μM decylubiquinone. The rates were corrected for the non-enzymatic reaction. After 1 min, the reaction was inhibited by adding various amounts (0.01-50 µM) of ND-011992. Each data point was assayed in triplicates. The standard deviation of each measurement is provided.
The duroquinol:oxygen oxidoreductase activity of isolated E. coli bd-I, bd-II and bo 3 oxidase was determined by monitoring the oxygen consumption with a Clark-type oxygen electrode (Oxyview System, Hansatech Instruments) 20,33 . All steps were conducted at 30 °C. The reaction chamber was filled with 2 mL 20 mM MOPS, 20 mM NaCl, 0.003% LMNG, pH 7.0. After formation of a stable baseline, 10 μL dithiothreitol (DTT) (1 M) and 5 μL duroquinone in ethanol (100 mM) were added and incubated for 1 min. The non-enzymatic reaction rate was determined. 10 μL enzyme in LMNG (2 mg/mL) were added to start the reaction. The rates were corrected for the non-enzymatic reaction. After 1 min, the reaction was inhibited by adding various amounts (0.01-50 µM) of ND-011992 and 2, respectively. Each data point was measured in triplicates. The standard deviation of each measurement is provided.
The NADH oxidase activity of isolated mitochondria from bovine heart was determined with a Clark-type oxygen electrode at 30 °C 34 . 50 µL mitochondrial membranes in 2 mL 0.44 M sucrose, 20 mM Tris, 2 mM EDTA, pH 7.4 were placed in the reaction chamber. After formation of a stable baseline, 20 µL NADH (0.5 M) was added to start the reaction. After 1 min, the reaction was inhibited by adding various amounts (0.01-50 µM) ND-011992. Each data point was assayed in triplicates. The standard deviation of each measurement is provided.

Data availability
The source data underlying Figs. 4, 5, 6 and other data are available from the corresponding authors upon reasonable request. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.